• Share
  • Email
  • Embed
  • Like
  • Save
  • Private Content
E coli lectur revised alpana
 

E coli lectur revised alpana

on

  • 3,113 views

 

Statistics

Views

Total Views
3,113
Views on SlideShare
3,113
Embed Views
0

Actions

Likes
1
Downloads
150
Comments
0

0 Embeds 0

No embeds

Accessibility

Upload Details

Uploaded via as Microsoft PowerPoint

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment
  • EMB is both selective and differential

E coli lectur revised alpana E coli lectur revised alpana Presentation Transcript

  • E .coli By DR Alpana verma
  • Introduction
    • The family Enterobacteriaceae is the largest, most heterogeneous collection of medically important gram-negative rods.
    • A total of more than 40 genera and 150 species and subspecies have been described.
    • These genera have been classified based on biochemical properties, antigenic structure, and nucleic acid hybridization and sequencing.
    • Despite the complexity of this family, almost 20 species are responsible for more than 95% of the infections .
    • Enterobacteriaceae are ubiquitous organisms, found worldwide in soil, water, and vegetation, and are part of the normal intestinal flora of most animals, including humans.
    • These bacteria cause a variety of human diseases, including 30% to 35% of all septicemias, more than 70% of urinary tract infections (UTIs), and many intestinal infections.
    • Some organisms (e.g., Salmonella typhi, Shigella species, Yersinia pestis ) are always associated with disease, whereas others (e.g., Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis ) are members of the normal commensal flora that can cause opportunistic infections.
  • Common Medically Important Enterobacteriaceae
    • Citrobacter freundii, Citrobacter koseri
    • Enterobacter aerogenes, Enterobacter cloacae
    • Escherichia coli
    • Klebsiella pneumoniae, Klebsiella oxytoca
    • Morganella morganii
    • Proteus mirabilis, Proteus vulgaris
    • Salmonella enterica
    • Serratia marcescens
    • Shigella sonnei, Shigella flexneri
    • Yersinia pestis, Yersinia enterocolitica,
    • Yersinia pseudotuberculosis
  • Figure 31-1 Sites of infections with common members of the Enterobacteriaceae listed in order of prevalence . © 2005 Elsevier
  • Physiology & Structure
    • Members of the Enterobacteriaceae family are moderately sized (0.3 to 1.0 × 1.0 to 6.0 μm) gram-negative rods .
    • They share a common antigen (enterobacterial common antigen), are either nonmotile or motile with peritrichous flagella), and do not form spores.
    • All members can grow rapidly, aerobically and anaerobically (facultative anaerobes), on a variety of nonselective (e.g., blood agar) and selective (e.g., MacConkey agar) media.
    • The Enterobacteriaceae have simple nutritional requirements, ferment glucose, reduce nitrate, and are catalase positive and oxidase negative.
    • The absence of cytochrome oxidase activity is an important characteristic, because it can be measured rapidly with a simple test and is used to distinguish the Enterobacteriaceae from many other fermentative and nonfermentative gram-negative rods.
    • A few exceptions to these rules exist (e.g., Plesiomonas shigelloides is oxidase positive; Klebsiella granulomatis cannot be cultured on traditional media).
    • Characteristics of the organisms' colonies on different media have been used to identify common members of the family Enterobacteriaceae.
  • Physiology & Structure
    • For example, the ability to ferment lactose has been used to differentiate lactose-fermenting strains (e.g., Escherichia, Klebsiella, Enterobacter, Citrobacter, and Serratia spp.) from strains that do not ferment lactose or do so slowly (e.g., Proteus, Salmonella, Shigella, and Yersinia spp.).
    • Resistance to bile salts in some selective media has been used to separate enteric pathogens (e.g., Shigella, Salmonella ) from commensal organisms that are inhibited by bile salts (e.g., gram-positive and some gram-negative bacteria present in the gastrointestinal tract).
    • Some Enterobacteriaceae have prominent capsules (e.g., Klebsiella, some Enterobacter and Escherichia strains),
    • The heat-stable lipopolysaccharide (LPS) is the major cell wall antigen and consists of three components: the outermost somatic O polysaccharide, a core polysaccharide common to all Enterobacteriaceae (enterobacterial common antigen), and lipid A
  • Antigenic structure of Enterobacteriaceae. Downloaded from: StudentConsult (on 19 July 2009 04:26 PM) © 2005 Elsevier
  • Escherichia coli
      • Normal inhabitant of the G.I. tract
      • Some strains cause various forms of gastroenteritis.
      • Is a major cause of urinary tract infection and neonatal meningitis and septicemia.
  • Morphology and culture characteristics
    • Morphology
      • Gram negative rods
      • Non-spore former, non-caspulated and motile
    • Culture characteristics
      • N.agar – circular, convex, small colonies
      • MacConkey medium – rose pink
      • Eosin Methylene blue – Metallic sheen colonies
  • Escherichia coli
    • The genus Escherichia consists of five species, of which E. coli is the most common and clinically most important.
    • This organism is associated with a variety of diseases, including sepsis, UTIs, meningitis, and gastroenteritis.
    • As expected, the multitude of strains capable of causing disease is reflected in the antigenic diversity of the bacteria.
    • Many O, H, and K antigens have been described, and they are used to classify the isolates for epidemiologic purposes.
    • Specific antigenic serogroups are also associated with greater virulence.
  • Diagnosis
    • Laboratory identification tests
  • Gram negative Rods of E.coli
  • Typical Peritrichous E.coli
  • MacConkey Agar plate Lactose fermentation
    • Biochemical reactions
    • IMViC: ++--
    • TSI: Acid butt/acid slant with gas production
    • • E. coli (pink/red) +
    • • E. coli (left side) –
    • • Kovac’s reagent
    • detects if tryptophan
    • has been hydrolyzed
    • to indol/tryptophanase
    Indole (IMViC tests)
  • Enterobacter aerogenes (left) – E. coli (bright red) + Reagent: Methyl red indicator identifies pH change due to mixed acid fermentation Methyl Red (MR) (IMViC tests)
  • Enterobacter aerogenes +(left) E. coli – (right) Barritt’s reagent Tests for acetoin, precursor to 2,3 butanediol fermentation Voges – Proskauer (VP) (IMViC tests)
    • • E. coli (left green) –
    • • Enterobacter aerogenes
    • (right royal blue) +
    • Reagent: Bromothymol
    • blue indicator
    • tests for ability to use citrate as
    • sole carbon source/citrate permease
    Citrate (IMViC tests)
    • Triple Sugar Iron Agar (TSI):
      • Purpose: To differentiate bacteria based on their ability to ferment glucose, lactose and/or sucrose, and to reduce sulfur to hydrogen sulfide.
  • INTERPRETATION OF TUBES ABOVE TUBE 1 (UNINOCULATED) TUBE 2 TUBE 3 TUBE 4 TUBE 5 SLANT - A A K K BUTT - A A A A HYDROGEN SULFIDE - - - + + GAS - - + - - A=Acidic K=Alkaline
  • Antigenic structure
    • Has O, H, and K antigens.
    • K1 has a strong association with virulence, particularly meningitis in neonates.
    • Antigenic structure .
    • Complex
    • Lipopolisaccharides/ Somatic or O antigen
      • Heat stable.more than 150 types
      • Most external in the cell wall
      • detected by bacterial agglutination
      • Antibody produced is predominantly IgM
    • Capsular/K antigen
      • Sometimes external to O antigen but not always
      • Can be polysaccharides or protein
    • Flagella /H antigen
      • Heat and alcohol labile.
    • Colicines/Bacteriocines
    • Produced by many Gram -ves
    • Virus like bactericidal substance
    • Active against some other bacteria of similar
    • or closely related species
  • E. coli toxins
      • Virulence factors
        • Toxins
          • Enterotoxins – produced by enterotoxigenic strains of E. coli (ETEC). Causes a movement of water and ions from the tissues to the bowel resulting in watery diarrhea. There are two types of enterotoxin:
            • LT – is heat labile and binds to specific Gm 1 gangliosides on the epithelial cells of the small intestine where it stimulates production of cAMP.
            • Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel.
  • E. coli toxins
            • ST – is heat stable and binds to specific receptors to stimulate the production of cGMP with the same results as with LT.
  • Clinical Diseases
    • Septicaemia
      • Typically, septicemia caused by gram-negative rods such as E. coli originates from infections in the urinary or gastrointestinal tract (e.g., intestinal perforation leading to an intraabdominal infection).
      • The mortality associated with E. coli septicemia is high for patients in whom immunity is compromised or the primary infection is in the abdomen or central nervous system (CNS).
    • Urinary Tract Infection
      • Most gram-negative rods that produce UTIs originate in the colon, contaminate the urethra, ascend into the bladder, and may migrate to the kidney or prostate.
      • Although most strains of E. coli can produce UTIs, disease is more common with certain specific serogroups.
      • These bacteria are particularly virulent because of their ability to produce adhesins), which bind to cells lining the bladder and upper urinary tract (preventing the elimination of the bacteria in voided urine), and hemolysin HlyA, which lyses erythrocytes and other cell types (leading to cytokine release and stimulation of an inflammatory response).
  • Clinical Diseases
    • Neonatal Meningitis
      • E. coli and group B streptococci cause the majority of CNS infections in infants younger than 1 month.
      • Approximately 75% of the E. coli strains possess the K1 capsular antigen.
      • This serogroup is also commonly present in the gastrointestinal tracts of pregnant women and newborn infants.
      • However, the reason this serogroup has a predilection for causing disease in newborns is not understood.
  • Clinical Diseases
    • Gastroenteritis
      • The strains of E. coli that cause gastroenteritis are subdivided into the following five major groups: enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), and enteroaggregative (EAEC) E. coli (.
        • EPEC
          • Enteropathogenic E. coli was the first E. coli associated with diarrheal disease and remains a major cause of infant diarrhea in impoverished countries.
          • Disease is rare in older children and adults, presumably because they have developed protective immunity.
          • Although specific O serogroups have been associated with outbreaks of EPEC diarrhea in nurseries, the serotyping of the E. coli isolated in random or endemic disease is discouraged except in epidemiologic investigations.
          • Disease is characterized by bacterial attachment to epithelial cells of the small intestine, with subsequent effacement (destruction) of the microvillus (attachment/effacement [A/E] histopathology).
          • The genes responsible for the "locus of enterocyte effacement (LEE)" reside on a pathogenicity island.
  • Clinical Diseases
          • This island of more than 40 genes mediates attachment and destruction of the host mucosal surface.
          • EPEC strains form microcolonies on the epithelial cell surface with the bacteria attached to the host cells by means of cuplike pedestals.
          • Initially a loose attachment mediated by bundle-forming pili occurs, followed by active secretion of proteins by the bacterial type III secretion system into the host epithelial cell.
          • One protein, translocated intimin receptor (Tir), is inserted into the epithelial cell membrane (this process is mediated by two other secreted proteins) and functions as a receptor for an outer membrane bacterial adhesin, intimin.
          • The watery diarrhea characteristic of this disease results from malabsorption caused by microvilli destruction.
        • ETEC
          • Disease caused by enterotoxigenic E. coli is seen most commonly in developing countries (an estimated 650 million cases per year), although almost 80,000 cases are estimated to occur annually in travelers from the United States.
          • Infections are observed in either young children in developing countries or travelers to these areas.
  • Gastroenteritis Caused by Escherichia coli A/E, Attachment/effacement. Organism Site of Action Disease Pathogenesis Enteropathogenic E. coli (EPEC) Small intestine Infant diarrhea in underdeveloped countries; watery diarrhea and vomiting, nonbloody stools Plasmid-mediated A/E histopathology with disruption of normal microvillus structure resulting in malabsorption and diarrhea Enterotoxigenic E. coli (ETEC) Small intestine Traveler's diarrhea; infant diarrhea in developing countries; watery diarrhea, vomiting, cramps, nausea, low-grade fever Plasmid-mediated, heat-stable and/or heat-labile enterotoxins that stimulate hypersecretion of fluids and electrolytes Enterohemorrhagic E. coli (EHEC) Large intestine Initial watery diarrhea, followed by grossly bloody diarrhea (hemorrhagic colitis) with abdominal cramps; little or no fever; may progress to hemolytic uremic syndrome (HUS) Mediated by cytotoxic Shiga toxins (Stx-1, Stx-2), which disrupt protein synthesis; A/E lesions with destruction of intestinal microvillus resulting in decreased absorption Enteroinvasive E. coli (EIEC) Large intestine Disease in underdeveloped countries; fever, cramping, watery diarrhea; may progress to dysentery with scant, bloody stools Plasmid-mediated invasion and destruction of epithelial cells lining colon Enteroaggregative E. coli (EAEC) Small intestine Infant diarrhea in underdeveloped countries; traveler's diarrhea; persistent watery diarrhea with vomiting, dehydration, and low- grade fever Plasmid-mediated aggregative adherence of rods ("stacked bricks") with shortening of microvilli, mononuclear infiltration, and hemorrhage; decreased fluid absorption
  • Clinical Diseases
          • The inoculum for disease is high, so infections are primarily acquired through consumption of fecally contaminated food or water.
          • Person-to-person spread does not occur.
          • ETEC produce two classes of enterotoxins: heat-labile toxins (LT-I, LT-II) and heat-stabile toxins (STa and STb).
          • Whereas LT-II is not associated with human disease, LT-I is functionally and structurally similar to cholera toxin (see Chapter 32 ) and is associated with human disease.
          • This toxin consists of one A subunit and five identical B subunits. The B subunits bind to the same receptor as cholera toxin (GM1 gangliosides), as well as other surface glycoproteins on epithelial cells in the small intestine.
          • After endocytosis, the A subunit of LT-I translocates across the membrane of the vacuole.
          • The A subunit has adenosine diphosphate (ADP)-ribosyltransferase activity and interacts with a membrane protein (Gs) that regulates adenylate cyclase.
  • Clinical Diseases
          • The net effect of this interaction is an increase in cyclic adenosine monophosphate (cAMP) levels, with enhanced secretion of chloride and a decreased absorption of sodium and chloride.
          • These changes are manifested in a watery diarrhea.
          • Exposure to the toxin also stimulates prostaglandin secretion and production of inflammatory cytokines, resulting in further fluid loss.
          • STa, but not STb, is associated with human disease.
          • STa is a small, monomeric peptide that binds to the transmembrane guanylate cyclase receptor, leading to an increase in the level of cyclic guanosine monophosphate and subsequent hypersecretion of fluids.
          • Genes for LT-I and STa are present on a transferable plasmid, which can also carry the genes for adhesins (CFA/I, CFA/II, CFA/III).
          • The colonization factors are fimbriae that recognize specific host glycoprotein receptors (define the host specificity). Both the toxin and colonization factors are required for disease to develop.
  • Clinical Diseases
          • Secretory diarrhea caused by ETEC develops after a 1- to 2-day incubation period and persists for an average of 3 to 4 days.
          • The symptoms (watery diarrhea and abdominal cramps; nausea and vomiting are less commonly observed) are similar to those of cholera but are milder.
          • Neither histologic changes of the intestinal mucosa nor inflammation is observed.
          • Disease mediated by heat-labile toxin is indistinguishable from that mediated by heat-stable toxin.
          • Toxin production is not associated with specific serogroups, so culture combined with immunoasays for the detection of the heat-labile and heat-stable toxins must be performed.
          • Commercial assays have been developed that detect toxin in cell cultures, but these tests are primarily used in reference labs.
  • Clinical Diseases
        • EHEC
          • Enterohemorrhagic E. coli strains are the most common strains producing disease in developed countries. It is estimated that these bacteria cause 73,000 infections and 60 deaths each year in the United States.
          • The ingestion of fewer than 100 bacteria can produce disease. The severity of the disease caused by EHEC ranges from mild, uncomplicated diarrhea to hemorrhagic colitis with severe abdominal pain, bloody diarrhea, and little or no fever.
          • More than 50 serogroups of EHEC have been isolated; however, the majority that cause human disease in the United States are believed to be serotype O157 : H7.
          • Hemolytic uremic syndrome (HUS), a disorder characterized by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia, is a complication in 5% to 10% of infected children younger than 10 years.
          • EHEC disease is most common in the warm months, and the highest incidence is in children younger than 5 years.
  • Clinical Diseases
          • Most cases of disease have been attributed to the consumption of undercooked ground beef or other meat products, water, unpasteurized milk or fruit juices (e.g., cider made from apples contaminated with feces from cattle), uncooked vegetables, and fruits.
          • Initially, a nonbloody diarrhea with abdominal pain develops in patients after 3 to 4 days of incubation.
          • Vomiting is observed in approximately half the patients.
          • Within 2 days of onset, disease in 30% to 65% of patients progresses to a bloody diarrhea with severe abdominal pain.
          • Complete resolution of symptoms typically occurs after 4 to 10 days in most untreated patients; however, HUS is a serious complication, particularly in young children.
          • Death can occur in 3% to 5% of patients with HUS, and severe sequelae (e.g., renal impairment, hypertension, CNS manifestations) can occur in as many as 30% of patients.
          • EHEC strains express a Shiga toxin (i.e., Stx-1, Stx-2, or both), induce A/E lesions on epithelial cells, and possess a 60-MDa plasmid that carries genes for other virulence factors.
  • Clinical Diseases
          • Stx-1 is essentially identical to the Shiga toxin produced by Shigella dysenteriae; Stx-2 has 60% homology.
          • Both toxins are acquired by lysogenic bacteriophages. Both have one A subunit and five B subunits, with the B subunits binding to a specific glycolipid on the host cell (globotriaosylceramide, GB3).
          • A high concentration of GB3 receptors is found in the intestinal villus and renal endothelial cells.
          • After the A subunit is internalized, it is cleaved into two molecules, and the A1 fragment binds to 28S ribosomal ribonucleic acid (rRNA) and disrupts protein synthesis.
          • Destruction of the intestinal villus results in decreased absorption with a relative increase in fluid secretion.
          • HUS has been preferentially associated with the production of Stx-2, which has been shown to destroy glomerular endothelial cells.
          • Damage to the endothelial cells leads to platelet activation and thrombin deposition, which in turn results in decreased glomerular filtration and acute renal failure.
  • Clinical Diseases
          • The Shiga toxins also stimulate expression of inflammatory cytokines (e.g., tumor necrosis factor [TNF]-α, interleukin [IL]-6), which among other effects enhance expression of GB3.
          • Two approaches have been used to detect EHEC: culture and toxin detection. In contrast with most E. coli, many O157 strains do not ferment sorbitol.
          • Sorbitol-containing MacConkey agar (S-MAC) has been used to screen stool specimens for sorbitol-negative (colorless), gram-negative bacteria that are then confirmed by serogrouping and biochemical tests to be O157 E. coli.
          • The limitations to this approach are some strains of O157 and many other EHEC serotypes ferment sorbitol and toxin production is not assessed.
          • The preferred method to detect EHEC is to culture stool specimens on nonselective MacConkey agar and then to assay isolated colonies for toxin production by commercially available immunoassays.
          • Unfortunately, the direct detection of toxin in stool samples is currently too insensitive to recommend.
  • Clinical Diseases
        • EIEC
          • Enteroinvasive E. coli strains are rare in the United States and uncommon in developing countries.
          • Pathogenic strains are primarily associated with a few restricted O serotypes: O124, O143, and O164.
          • The strains are closely related by phenotypic and pathogenic properties to Shigella.
          • The bacteria are able to invade and destroy the colonic epithelium, producing a disease characterized initially by watery diarrhea.
          • A minority of patients progress to the dysenteric form of disease, consisting of fever, abdominal cramps, and blood and leukocytes in stool specimens.
          • A series of bacterial genes carried on a plasmid mediate invasion ( pInv genes) into the colonic epithelium.
          • The bacteria then lyse the phagocytic vacuole and replicate in the cell cytoplasm.
          • Movement within the cytoplasm and into adjacent epithelial cells is regulated by formation of actin tails (similar to that observed with Listeria ).
  • Clinical Diseases
          • This process of epithelial cell destruction with inflammatory infiltration can progress to colonic ulceration.
          • Detection of EIEC strains is restricted to research laboratories.
          • Although immunoassays and nucleic acid based assays have been developed for detecting invasion-related factors, the usefulness of these assays is limited by the fact the genes for these virulence factors reside on a large plasmid that is rapidly lost in vitro.
        • EAEC
          • Enteroaggregative E. coli strains have been implicated as a cause of persistent, watery diarrhea with dehydration in infants in developing countries and in travelers to these countries.
          • The persistence of these bacteria is associated with chronic diarrhea and growth retardation in children.
          • The bacteria are characterized by their autoagglutination in a "stacked-brick" arrangement.
          • This process is mediated by bundle-forming fimbriae (aggregative adherence fimbriae I and II), which are carried on a plasmid.
  • Clinical Diseases
          • EAEC stimulate secretion of mucus, which traps the bacteria in a biofilm overlying the epithelium of the small intestine. Shortening of the microvilli, mononuclear infiltration, and hemorrhage are then observed.
          • A cytotoxin has not been demonstrated but is likely to be present. Diagnosis of these infections is primarily restricted to research laboratories.
  • Treatment of E.coli related diarrhoea
    • 1st Line
    • Nitrofurantoin
    • Nalidixic acid
    • Norfloxacin
    • Ampicillin
    • Cotrimoxazole
    • 2nd line
    • Ciprofloxacin/Ceftriaxone/Cefuroxime
    • Gentamicin
  • Summary of Escherichia coli
    • Physiology and Structure
      • Gram-negative, facultative anaerobic rods
      • Fermenter; oxidase negative
      • Outer membrane makes the organisms susceptible to drying
      • Lipopolysaccharide consists of outer somatic O polysaccharide, core polysaccharide (common antigen), and lipid A (endotoxin)
    • Virulence
      • Refer to Boxes 31-2 and 31-3
      • Endotoxin
      • Permeability barrier of outer membrane
      • Adhesins (e.g., colonization factor antigen, Dr adhesins)
      • Exotoxins (e.g., heat-stabile and heat-labile enterotoxins, Shiga toxins)
      • Invasive capacity
  • Summary of Escherichia coli
    • Epidemiology
      • Most common aerobic, gram-negative rods in the gastrointestinal tract
      • Most infections are endogenous (patient's normal microbial flora)
      • Strains causing gastroenteritis are generally acquired exogenously
    • Diseases
      • Bacteremia (most commonly isolated gram-negative rod in the United States)
      • Urinary tract infection (most common cause of bacterial UTIs); limited to bladder (cystitis) or can spread to kidneys (pyelonephritis) or prostate (prostatitis)
      • At least five different pathogenic groups cause gastroenteritis (EPEC, ETEC, EHEC, EIEC, EAEC); most cause diseases in developing countries, although EHEC is an important cause of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in the United States
      • Neonatal meningitis (usually with strains carrying the K1 capsular antigen)
      • Intraabdominal infections (associated with intestinal perforation)
  • Summary of Escherichia coli
    • Diagnosis
      • Organisms grow rapidly on most culture media
      • Enteric pathogens with the exception of EHEC are detected only in reference or research laboratories
    • Treatment, Prevention, and Control
      • Enteric pathogens are treated symptomatically unless disseminated disease occurs
      • Antibiotic therapy is guided by in vitro susceptibility tests
      • Appropriate infection-control practices are used to reduce the risk of nosocomial infections (e.g., restricting use of antibiotics, avoiding unnecessary use of urinary tract catheters)
      • Maintenance of high hygienic standards to reduce the risk of exposure to gastroenteritis strains
      • Proper cooking of beef products to reduce risk of EHEC infections